CONSTITUTIVE ACTIVATION OF THE BETA(2) ADRENERGIC-RECEPTOR ALTERS THEORIENTATION OF ITS 6TH MEMBRANE-SPANNING SEGMENT

The binding site of the beta(2) adrenergic receptor, like that of othe r homologous G-protein-coupled receptors, is contained within a water- accessible crevice formed among its seven membrane spanning segments. Methanethiosulfonate ethylammonium (MTSEA), a charged, hydrophilic, li pophobic, sulfhydryl-specific reagent, had no effect on the binding of agonist or antagonist to wild type beta(2) receptor expressed in HEK 293 cells. This suggested that no endogenous cysteines are accessible in the binding site crevice. In contrast, in a constitutively active b eta(2) receptor, MTSEA significantly inhibited antagonist binding, and isoproterenol slowed the rate of reaction of MTSEA. This implies that at least one endogenous cysteine becomes accessible in the binding si te crevice of the constitutively active beta(2) receptor, Cys-285, in the sixth membrane-spanning segment, is responsible for the inhibitory effect of MTSEA on ligand binding to the constitutively active mutant . The acquired accessibility of Cys-285 in the constitutively active m utant may result from a rotation and/or tilting of the sixth membrane- spanning segment associated with activation of the receptor. This rear rangement could bring Cys-285 to the margin of the binding site crevic e where it becomes accessible to MTSEA.